A method for preparing ultrathin cell sections and its application

Abstract

This invention relates to the field of biotechnology, particularly to the field of IPC G01N1, and further to a method for preparing and applying ultrathin cell sections. The method includes the following steps: (1) sample collection; (2) pre-fixation; (3) agar pre-embedding; (4) fixation; (5) dehydration, permeation, and embedding; (6) ultrathin sectioning; and (7) staining. This invention uses tannic acid instead of osmium tetroxide for sample fixation, avoiding the harm of osmium tetroxide to human health and reducing costs. During tannic acid fixation, uranium acetate is added for further fixation, which avoids incomplete cell membranes, prevents the formation of large amounts of cell debris, and prevents cell swelling, further improving the subsequent staining effect, especially suitable for the fixation of CHO cells.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, particularly to the field of IPC G01N1, and further to a method for preparing ultrathin cell sections and their application. Background Technology

[0002] Transmission electron microscopy (TEM) is primarily used to observe the fine internal structures of cells. Because electrons have weak penetrating power, specimens for TEM observation must be cut into ultrathin sections of approximately 50 nm. Before ultrathin sectioning, samples must undergo fixation, dehydration, and embedding. After sectioning, they must also undergo staining before electron microscopy observation. Traditional cell ultrathin section preparation requires fixation with osmium tetroxide (OST), a highly toxic chemical; moderate doses can be fatal. According to the "Regulations on the Safety Management of Hazardous Chemicals," the purchase, transportation, storage, and use of OST are strictly regulated by public security departments, resulting in high management costs. For example, the study by Li Xinhui et al. in *Progress in Biochemistry and Biophysics*, which investigated the effect of different thicknesses of cell thin sections on the contrast of AFM images, disclosed a sample preparation method using 1% OST fixative to fix cells. Summary of the Invention

[0003] To solve the above-mentioned technical problems, the first aspect of the present invention provides a method for preparing ultrathin cell sections, comprising the following steps: (1) sample collection; (2) pre-fixation; (3) agar pre-embedding; (4) fixation; (5) dehydration, permeation, and embedding; (6) ultrathin sectioning; and (7) staining.

[0004] Preferably, the fixative in step (2) is selected from any one of glutaraldehyde solution, formaldehyde solution, methanol solution, ethanol solution, and paraformaldehyde solution.

[0005] Preferably, the fixative in step (2) is a glutaraldehyde solution.

[0006] Preferably, the solvent used to prepare the glutaraldehyde solution is PB buffer (phosphate buffer), PBS buffer (phosphate buffer), or physiological saline.

[0007] Preferably, the solvent used to prepare the glutaraldehyde solution is PB buffer solution.

[0008] Preferably, the concentration of the PB buffer solution is 0.05–0.2 mol / L.

[0009] Preferably, the concentration of the PB buffer solution is 0.1 mol / L.

[0010] Preferably, the specific method for pre-fixation in step (2) is as follows: fixation with glutaraldehyde solution at a temperature of 2-8°C for 10-15 hours.

[0011] Preferably, the volume concentration of the glutaraldehyde solution is 2-4%.

[0012] Preferably, the specific method for agar pre-embedding in step (3) is as follows: prepare an agarose aqueous solution, mix the agarose aqueous solution with the sample evenly, cool and solidify, and then wash with 0.1mol / L PBS buffer 3 to 5 times, each time for 5 to 15 minutes.

[0013] Preferably, the mass concentration of the agarose aqueous solution is 1-3%.

[0014] Preferably, the fixation process in step (4) uses a tannic acid solution for fixation.

[0015] Preferably, the solvent used to prepare the tannic acid solution is water, acetone, PB buffer, PBS buffer, sodium dimethylarsinate buffer, or physiological saline.

[0016] Preferably, the solvent used to prepare the tannic acid solution is a mixture of water, ethanol and acetone.

[0017] Preferably, the volume ratio of water, ethanol and acetone is (60-70):(30-40):1.

[0018] Preferably, the volume ratio of water, ethanol and acetone is 65:34:1.

[0019] Preferably, the specific method for fixing the tannic acid solution is as follows: fix with tannic acid solution at a temperature of 22-25℃ for 1-4 hours, and then wash with 0.1mol / L PB buffer 3-5 times, each time for 5-15 minutes.

[0020] Preferably, the mass concentration of the tannic acid solution is 0.5% to 2%.

[0021] Preferably, the mass concentration of the tannic acid solution is 1%.

[0022] Preferably, the specific method for fixation in step (4) is as follows: fixation is performed for 2.5 h using tannic acid solution at a temperature of 22-25°C, followed by rinsing with 0.1 mol / L PB buffer 4 times, 10 min each time.

[0023] Preferably, the fixation process in step (4) further includes fixation using a uranium acetate solution.

[0024] Preferably, the solvent used in preparing the uranium acetate-hydrogen peroxide solution is acetone.

[0025] Preferably, the specific method for fixing with uranium acetate solution is as follows: fix with uranium acetate solution at a temperature of 2-8°C for 1-3 hours, and then wash with 0.1 mol / L PB buffer 3-5 times, each time for 5-15 minutes.

[0026] Preferably, the mass concentration of the uranium acetate solution is 1% to 3%.

[0027] Preferably, the mass concentration of the uranium acetate solution is 2%.

[0028] Preferably, the specific method for fixing with uranium acetate solution is as follows: fix with uranium acetate solution at a temperature of 4°C for 2 hours, and then wash with 0.1 mol / L PB buffer 4 times, 10 minutes each time.

[0029] This invention uses tannic acid to fix samples. Tannic acid is a secondary product of plant metabolism during plant growth and is a type of water-soluble polyphenol compound. Tannic acid can fix many proteins, carbohydrate derivatives, polypeptides, and saturated phospholipids. However, during the experiment, the applicant found that using only tannic acid for fixation sometimes resulted in unclear cell edges and blurry images in ultrathin cell sections after staining. The applicant unexpectedly discovered that when uranium acetate was added during the tannic acid fixation process for further fixation, and the tannic acid solution concentration was controlled at 1% for 3 hours, and the acetone-based uranium acetate solution concentration was 2% for 2 hours, it could avoid cell membrane incompleteness, prevent the formation of large amounts of cell debris, and prevent cell swelling. Furthermore, it could improve the subsequent staining effect, especially for CHO cells (Chinese hamster ovary cells). The applicant hypothesizes that because tannic acid has a large molecular weight, its permeability to cells is poor, and using tannic acid for cell fixation may lead to uneven cell fixation and poor results. When uranium acetate is used for fixation after tannic acid fixation, the uranium acetate can react with phosphate groups, as well as acidic and basic groups in proteins, thereby further fixing the cells. By controlling the mass concentrations of tannic acid and uranium acetate, as well as the fixation time, the overall performance of ultrathin sections of cells can be improved.

[0030] Preferably, the specific method for dehydration in step (5) is as follows: first, dehydration is carried out using an ethanol aqueous solution with a volume percentage of 30-90%, and then dehydration is carried out using an acetone aqueous solution with a volume percentage of 90-100%.

[0031] Preferably, the specific method for dehydration in step (5) is as follows: using an ethanol aqueous solution with a volume percentage of 30%, 50%, 70%, and 90% to dehydrate for 10-15 minutes each, and then using an acetone aqueous solution with a volume percentage of 90% and 100% to dehydrate for 15-20 minutes each.

[0032] Preferably, the specific method of permeation in step (5) is to use acetone:resin = 2:(1-4) for permeation.

[0033] Preferably, the specific method of permeation in step (5) is as follows: using a solution of acetone:resin = 2:1, 1:1, or 1:2, and permeating for 0.5-1.5h, 1.5-2.5h, or 1.5-2.5h respectively.

[0034] Preferably, the resin is selected from any one of SPon812 epoxy resin, Epon812 epoxy resin, Spurr epoxy resin, TAAB812 epoxy resin, and epoxy resin 618.

[0035] Preferably, the resin is Epon812 epoxy resin.

[0036] Preferably, the specific method of embedding in step (5) is as follows: embedding with Epon812 epoxy resin, embedding at 40-50℃ for 18-26h and embedding at 55-60℃ for 18-26h respectively.

[0037] Preferably, the thickness of the ultrathin slice in step (6) is 50-100 nm.

[0038] Preferably, the specific method of staining in step (7) is as follows: first, stain with uranium acetate for 15-25 minutes, rinse with deionized water; then, stain with lead citrate for 5-15 minutes, rinse with deionized water, and blot dry with filter paper.

[0039] The second aspect of the present invention provides an application of the above-described method for preparing ultrathin cell sections for transmission electron microscopy observation of cells.

[0040] Beneficial effects: This invention uses tannic acid instead of osmium tetroxide to fix samples, avoiding the harm of osmium tetroxide to human health and reducing costs. When uranium acetate is added for fixation again during the tannic acid fixation process, and the mass concentration of the tannic acid solution is controlled at 1% and the fixation time is 3 hours, while the mass concentration of the uranium acetate solution prepared with acetone is 2% and the fixation time is 2 hours, it can avoid cell membrane incompleteness, avoid the formation of a large number of cell fragments between cells, avoid cell swelling, and further improve the staining effect of subsequent cells, especially suitable for the fixation of CHO cells. Attached Figure Description

[0041] Figure 1 Transmission electron microscope images of ultrathin sections of CHO cells prepared for Example 1.

[0042] Figure 2 Transmission electron microscope images of ultrathin sections of CHO cells prepared for Example 2.

[0043] Figure 3 Transmission electron microscope images of ultrathin sections of CHO cells prepared for Example 3. Detailed Implementation

[0044] Example 1

[0045] Example 1 provides a method for preparing ultrathin cell sections, including the following steps: (1) sample collection; (2) pre-fixation; (3) agar pre-embedding; (4) fixation; (5) dehydration, permeation, and embedding; (6) ultrathin sectioning; (7) staining.

[0046] The sample was CHO cells.

[0047] The fixative used in step (2) is glutaraldehyde solution.

[0048] The solvent used to prepare the glutaraldehyde solution is PB buffer solution.

[0049] The specific method for pre-fixation in step (2) is as follows: fixation is performed for 13 hours using a glutaraldehyde solution with a volume concentration of 2.5% at a temperature of 6°C.

[0050] The concentration of the PB buffer solution is 0.1 mol / L.

[0051] The specific method for agar pre-embedding in step (3) is as follows: prepare an agarose aqueous solution, mix the agarose aqueous solution with the sample evenly, cool and solidify, and then rinse with PB buffer 4 times, 10 min each time.

[0052] The agarose aqueous solution has a mass concentration of 2%.

[0053] In step (4), the fixation process uses tannic acid solution for fixation.

[0054] The solvent used to prepare the tannic acid solution is a mixture of water, ethanol, and acetone, with a volume ratio of 65:34:1.

[0055] The mass concentration of the tannic acid solution is 1%.

[0056] The specific method for fixation in step (4) is as follows: fixation is performed for 2.5 h using tannic acid solution at a temperature of X℃, followed by rinsing with 0.1 mol / L PB buffer 4 times, 10 min each time.

[0057] The fixation process in step (4) also includes fixation using uranium acetate solution.

[0058] The solvent used in preparing the uranium acetate solution is acetone.

[0059] The mass concentration of the uranium acetate solution is 2%.

[0060] The specific method for fixing the uranium acetate solution is as follows: fix with uranium acetate solution at a temperature of 4°C for 2 hours, and then wash with 0.1 mol / L PB buffer 4 times, 10 minutes each time.

[0061] The specific method for dehydration in step (5) is as follows: use an ethanol aqueous solution with a volume percentage of 30%, an ethanol aqueous solution with a volume percentage of 50%, an ethanol aqueous solution with a volume percentage of 70%, and an ethanol aqueous solution with a volume percentage of 90% for 12 minutes each, and then use an acetone aqueous solution with a volume percentage of 90% and 100% acetone for 15 minutes each.

[0062] The specific method for permeation in step (5) is as follows: using solutions of acetone:resin = 2:1, 1:1, and 1:2, and permeating for 1 hour, 2 hours, 2 hours, and 2 hours respectively.

[0063] The resin is Epon812 epoxy resin.

[0064] The specific method for embedding in step (5) is as follows: Epon812 epoxy resin is used for embedding, and the embedding is carried out at 45°C for 24 hours and at 60°C for 24 hours respectively.

[0065] The thickness of the ultrathin slice in step (6) is 70 nm.

[0066] The specific method of staining in step (7) is as follows: first, stain with uranium acetate for 20 minutes, then rinse with deionized water; then stain with lead citrate for 10 minutes, rinse with deionized water, and dry with filter paper.

[0067] Example 2

[0068] Example 2 provides a method for preparing ultrathin cell sections, the specific implementation of which is the same as that of Example 1, except that the fixation process in step (4) does not include fixation with uranium acetate solution.

[0069] Example 3

[0070] Example 3 provides a method for preparing ultrathin cell sections, the specific implementation of which is the same as that of Example 1, except that the mass concentration of the tannic acid solution is 3%.

[0071] Performance testing: The ultrathin cell sections prepared in Examples 1-3 were observed using an HT-7700 transmission electron microscope. The test results are shown in [Figure number missing]. Figures 1-3 .

Claims

1. A method for preparing ultrathin cell sections, comprising the following steps: (1) Sample collection; (2) Pre-fixation; (3) Agar pre-embedding; (4) Fixation; (5) Dehydration, permeation, and embedding; (6) Ultrathin sectioning; (7) Staining; The fixation process in step (4) uses tannic acid solution for fixation; The specific method of fixation with tannic acid solution is: fix with tannic acid solution at a temperature of 22~25℃ for 1~4h, and then rinse with 0.1mol / L PB buffer 3~5 times, each time for 5~15min; The fixation process in step (4) also includes fixation with uranium acetate solution; The specific method of fixation with uranium acetate solution is: fix with uranium acetate solution at a temperature of 2~8℃ for 1~3h, and then rinse with 0.1mol / L PB buffer 3~5 times, each time for 5~15min.

2. The method for preparing ultrathin cell sections as described in claim 1, characterized in that, The specific method for agar pre-embedding in step (3) is as follows: prepare an agarose aqueous solution, mix the agarose aqueous solution with the sample evenly, cool and solidify, and then wash with 0.1mol / L PB buffer 3 to 5 times, each time for 5 to 15 minutes.

3. The method for preparing ultrathin cell sections as described in claim 1, characterized in that, The specific method of permeation in step (5) is as follows: acetone:resin = 2:(1~4) is used for permeation.

4. The method for preparing ultrathin cell sections as described in claim 3, characterized in that, The resin is selected from any one of SPon812 epoxy resin, Epon812 epoxy resin, Spurr epoxy resin, TAAB812 epoxy resin, and epoxy resin 618.

5. The method for preparing ultrathin cell sections as described in claim 1, characterized in that, The specific method of staining in step (7) is as follows: first, stain with uranium acetate for 15-25 minutes, then rinse with deionized water; then stain with lead citrate for 5-15 minutes, rinse with deionized water, and dry with filter paper.

6. The application of a method for preparing ultrathin cell sections as described in any one of claims 1-5, characterized in that, Used for transmission electron microscopy observation of cells.

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